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1.
Mechanism of cellobiose epimerase 总被引:3,自引:0,他引:3
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分别考察C.acetobutylicum 810705、810706以不同浓度的麸皮和玉米粉添加物作为营养元素,纤维二糖直接进行丙酮丁醇(ABE)发酵的结果,发现2株菌对于玉米粉和麸皮的浓度变化趋势一致,C.acetobutylicum 810706转化率较高。纤维二糖ABE发酵工艺条件表明:玉米粉添加量为总糖含量的30%、底物糖质量浓度60 g/L,pH 6.5、温度35℃时,C.acetobutylicum 810706转化率达到37.38%,总溶剂质量浓度22.43 g/L,比葡萄糖、木糖ABE发酵转化率高。模拟纤维素酶水解产物配制混合糖培养基,其溶剂转化率较单独的葡萄糖、木糖发酵的转化率高,为34.95%。对比纤维素酶水解条件,C.acetobutylicum 810706具有优良的纤维素酶水解同步糖化ABE发酵能力。 相似文献
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Recent advances in fungal cellobiose oxidoreductases 总被引:2,自引:0,他引:2
Karl-Erik L. Eriksson Naoto Habu Masahiro Samejima 《Enzyme and microbial technology》1993,15(12):1002-1008
When grown on cellulose, the white-rot fungus Phanerochaete chrysosporium (Sporotrichum pulverulentum), produces two cellobiose oxidoreductases, i.e., cellobiose:quinone oxidoreductase (CBQ) and cellobiose oxidase (CBO). Similar cellobiose-oxidizing enzymes, capable of utilizing a wide variety of electron acceptors, have been detected in many other fungi. However, the role of the cellobiose oxidoreductases in white-rot fungi, or in any fungi for that matter, is still not known. The original role ascribed to CBQ was as a link between cellulose and lignin degradation. CBQ has been shown to reduce quinones and phenoxyradicals released during lignin degradation concomitantly oxidizing cellobiose and other cellodextrins released during cellulose degradation. Thus, one function proposed for the cellobiose oxidoreductases is to prevent repolymerization of phenoxyradicals formed when phenoloxidases (peroxidases and laccases) attack lignin and lignin degradation products. However, evidence obtained so far indicates that the presence of CBO/CBQ with lignin peroxidases and laccases actually reduces the rate of oxidation of lignin degradation products. CBQ has a molecular mass of about 60 kD and contains an FAD cofactor. CBO contains both heme and FAD, and has a mass of about 90 kD. It has recently been demonstrated that CBO can be proteolytically cleaved into FAD and heme domains. The FAD domain of CBO seems to have all the properties of CBQ, suggesting that CBQ is a cleavage product of CBO. Whether CBO is a precursor of CBQ is not yet known. CBO and CBQ can be distinguished not only by the differences in their spectral properties, but also by the ability of CBO, but not CBQ, to reduce cytochrome c. Both CBO and CBQ have a cellulose-binding domain (CBD), as do a large number of endoglucanases and cellobiohydrolases. The induction-repression patterns regulating cellobiose oxidoreductase genes are not known in any detail. Most reports point to induction during cellulose degradation, but repression has not been studied. Induction has also been suggested to occur by addition of lignosulfonate to the medium. 相似文献
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Fridman V Wollenberger U Bogdanovskaya V Lisdat F Ruzgas T Lindgren A Gorton L Scheller FW 《Biochemical Society transactions》2000,28(2):63-70
An important aspect of the cytochrome c electrochemistry is the possibility of coupling the 'heterogeneous reactions' with other redox enzymes. Cellobiose dehydrogenase, a 89170 Da glycoprotein that contains both FAD and a b-type haem as prosthetic groups, donates electrons to a number of acceptors, including cytochrome c. While haem b is surrounded mainly by acidic amino acids, cytochrome c displays positive charged lysine groups around the haem site. Thus a fast reaction between both proteins is explicable. In the presence of cellobiose, a catalytic current was observed, owing to the interaction of cellobiose dehydrogenase with electrostatically adsorbed cytochrome c. Adsorption of cytochrome c provides a technological model surface for vectorial electron transfer. 相似文献
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The thermal transformations and rearrangements of β-cellobiose and trehalose have been investigated by dynamic thermal analysis and parallel, isothermal, chemical methods. The reducing disaccharide showed concurrent melting and thermal anomerization, followed by condensation and ultimate decomposition. The non-reducing disaccharide showed dehydration, melting, polymerization, and decomposition. The polymeric materials formed on isothermal heating of cellobiose and trehalose were shown to be randomly linked glucans containing pyranoid and furanoid rings and unsaturated components. 相似文献
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Anaerobic bacteria assimilate cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular β-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (ΔG°=+3.6 kJ mol−1). A thermodynamic “push” from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae. 相似文献
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Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by various wood-degrading fungi. It oxidizes soluble cellodextrins, mannodextrins and lactose efficiently to their corresponding lactones by a ping-pong mechanism using a wide spectrum of electron acceptors including quinones, phenoxyradicals, Fe(3+), Cu(2+) and triiodide ion. Monosaccharides, maltose and molecular oxygen are poor substrates. CDH that adsorbs strongly and specifically to cellulose carries two prosthetic groups; namely, an FAD and a heme in two different domains that can be separated after limited proteolysis. The FAD-containing fragment carries all known catalytic and cellulose binding properties. One-electron acceptors, like ferricyanide, cytochrome c and phenoxy radicals, are, however, reduced more slowly by the FAD-fragment than by the intact enzyme, suggesting that the function of the heme group is to facilitate one-electron transfer. Non-heme forms of CDH have been found in the culture filtrate of some fungi (probably due to the action of fungal proteases) and were for a long time believed to represent a separate enzyme (cellobiose:quinone oxidoreductase, CBQ). The amino acid sequence of CDH has been determined and no significant homology with other proteins was detected for the heme domain. The FAD-domain sequence belongs to the GMC oxidoreductase family that includes, among others, Aspergillus niger glucose oxidase. The homology is most distinct in regions that correspond to the FAD-binding domain in glucose oxidase. A cellulose-binding domain of the fungal type is present in CDH from Myceliophtore thermophila (Sporotrichum thermophile), but in others an internal sequence rich in aromatic amino acid residues has been suggested to be responsible for the cellulose binding. The biological function of CDH is not fully understood, but recent results support a hydroxyl radical-generating mechanism whereby the radical can degrade and modify cellulose, hemicellulose and lignin. CDH has found technical use in highly selective amperometric biosensors and several other applications have been suggested. 相似文献
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G Canevascini 《Analytical biochemistry》1985,147(2):419-427
An assay for cellulase activity based on the oxidation of cellobiose, formed during the cellulase reaction, with ferricyanide and a cellobiose dehydrogenase derived from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile is presented. Due to the restricted specificity of this enzyme for cellobiose and cellodextrins, glucose, which may be formed by the action of some cellulolytic components or by beta-glucosidase, does not contribute to the result. The negative interference of beta-glucosidase may be eliminated by glucono-delta-lactone inhibition. The assay, which is not influenced by cellobiose back-inhibition of the cellulase reaction, like the usual cellulase tests based on the increase in reducing power, is basically unspecific with respect to endo- or exo-acting enzymes giving rise to a total cellulase activity. With the use of an amorphous cellulose substrate (reprecipitated cellulose after dissolving in concentrated phosphoric acid), unpredictable effects due to cooperativity between endo- and exo-enzyme components were eliminated. An analytical procedure giving a linear response between activity and enzyme concentration and between activity and time of incubation has been worked out. 相似文献
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Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis. However, the general application of wild-type CP for glycoside synthesis is hampered by its relatively narrow substrate specificity. Recently we have taken some successful efforts to broaden the substrate specificity of Cellulomonas uda CP by directed evolution and protein engineering. This review will give an overview of the obtained results and address the applicability of the engineered CP enzymes for glycoside synthesis. CP is the first example of an extensively engineered disaccharide phosphorylase, and may provide valuable information for protein engineering of other phosphorylase enzymes. 相似文献
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Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis. However, the general application of wild-type CP for glycoside synthesis is hampered by its relatively narrow substrate specificity. Recently we have taken some successful efforts to broaden the substrate specificity of Cellulomonas uda CP by directed evolution and protein engineering. This review will give an overview of the obtained results and address the applicability of the engineered CP enzymes for glycoside synthesis. CP is the first example of an extensively engineered disaccharide phosphorylase, and may provide valuable information for protein engineering of other phosphorylase enzymes. 相似文献
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A pressurized, stirred vessel attached with an ultrafiltration membrane was used as a membrane reactor, Cellobiose hydrolysis by cellobiase was carried out and theoretically analyzed in terms of steady-state conversion and flow rate through the membrane. When the flow rate exceeds a critical value, a significant fraction of the enzyme inside the reactor is localized in the concentration polarization layer where shear from stirring is high. Consequently, enzyme deactivation inside the concentration polarization layer is accelerated and the conversion decreases due to an exchange of active enzyme in bulk with deactivated enzyme in the polarization layer via convection and back diffusion. Successful operation can be obtained at flow rates lower than the critical point to avoid the polarization and thus the deactivation. It is shown that 6.5 L of 2 mg/mL of cellobiose solution is hydrolyzed to glucose with a conversion of 91% in 20 h with 1.617 mg of cellobiase enzyme, in a reactor attached with a PM 10 membrane of an effective surface area of 39.2 cm2. 相似文献
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The utilization of cellobiose by Verticillium albo-atrum 总被引:1,自引:0,他引:1
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A comparison of the catalytic properties of cellobiose:quinone oxidoreductase and cellobiose oxidase from Phanerochaete chrysosporium. 总被引:3,自引:0,他引:3
Several catalytic properties of the FAD enzyme cellobiose:quinone oxidoreductase (CBQ) and the heme/FAD enzyme, cellobiose oxidase (CBO) have been investigated and compared. Dichlorophenol-indophenol was found to be a very good electron acceptor for cellobiose oxidation by both enzymes. The optimal pH value for this oxidation with dichlorophenol-indophenol as a co-substrate was observed around pH 4 for both enzymes. The turnover numbers of this reaction were also very similar. The Km values for cellobiose oxidation were identical, whereas the Km for CBO with dichlorophenol-indophenol is lower than that of CBQ. Atmospheric oxygen is a very poor electron acceptor for both CBO and CBQ, however, CBO can utilize cytochrome c as an effective electron acceptor, while CBQ cannot. The specific activity of CBO for cytochrome c is thus about 200-times higher than for oxygen. Thus, one way to distinguish the two enzymes is by the cytochrome-c-reducing ability of CBO. Therefore, we propose that the nomenclature for CBO is tentatively changed to cellobiose:cytochrome c oxidoreductase until a rational name can be installed. Both enzymes have radical-reducing activities. The cation radical, derived from 1,2,4,5-tetramethoxybenzene, was reduced by both enzymes at almost the same reaction rate. The phenoxyradical produced by lignin peroxidase, catalyzing the oxidation of acetosyringon, was also reduced by both enzymes. The reduction of phenoxyradicals formed by phenoloxidases (lignin peroxidases, as well as laccases) may be important in preventing repolymerization reactions which we suggest would significantly facilitate lignin degradation. 相似文献
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AYERS WA 《Journal of bacteriology》1958,76(5):515-517
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The cellobiose phosphorylase (EC 2.4.1.20) of Cellvibrio gilvus, which is an endocellular enzyme, has been purified 196-fold with a recovery of 11% and a specific activity of 27.4 mumol of glucose 1-phosphate formed/min per mg of protein. The purification procedure includes fractionation with protamine sulphate, and hydroxyapatite and DEAE-Sephadex A-50 chromatography. The enzyme appears homogeneous on polyacrylamide-gel electrophoresis, and a molecular weight of 280 000 was determined by molecular-sieve chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a single band and mol.wt. 72 000, indicating that cellobiose phosphorylase consists of four subunits. The enzyme had a specificity for cellobiose, requiring Pi and Mg2+ for phosphorylation, but not for cellodextrin, gentibiose, laminaribiose, lactose, maltose, kojibiose and sucrose. The enzyme showed low thermostability, an optimum pH of 7.6 and a high stability in the presence of 2-mercaptoethanol or dithiothreitol. The Km values for cellobiose and Pi were 1.25 mM and 0.77 mM respectively. Nojirimycin acted as a powerful pure competitive inhibitor (with respect to cellobiose) of the enzyme (Ki = 45 microM). Addition of thiol-blocking agents to the enzyme caused 56% inhibition at 500 microM-N-ethylmaleimide and 100% at 20 microM-p-chloromercuribenzoate. 相似文献
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Shuchi H. Desai Christine A. Rabinovitch-Deere Yohei Tashiro Shota Atsumi 《Applied microbiology and biotechnology》2014,98(8):3727-3736
Converting lignocellulosics into biofuels remains a promising route for biofuel production. To facilitate strain development for specificity and productivity of cellulosic biofuel production, a user friendly Escherichia coli host was engineered to produce isobutanol, a drop-in biofuel candidate, from cellobiose. A beta-glucosidase was expressed extracellularly by either excretion into the media, or anchoring to the cell membrane. The excretion system allowed for E. coli to grow with cellobiose as a sole carbon source at rates comparable to those with glucose. The system was then combined with isobutanol production genes in three different configurations to determine whether gene arrangement affected isobutanol production. The most productive strain converted cellobiose to isobutanol in titers of 7.64?±?0.19 g/L with a productivity of 0.16 g/L/h. These results demonstrate that efficient cellobiose degradation and isobutanol production can be achieved by a single organism, and provide insight for optimization of strains for future use in a consolidated bioprocessing system for renewable production of isobutanol. 相似文献
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Summary The metabolism ofBacteroides cellulosolvens was studied on cellobiose and cellulose as energy and carbon sources. The growth rate was faster on cellobiose; however, growth on cellulose resulted in consumption of 55% more hexose equivalents, and in production of 49% more biomass, and 30% more metabolites (ethanol, acetate, and lactate). On each substrateB. cellulosolvens exhibited two distinct ranges of molar growth yields (Y
H g cells/mol hexose). At low substrate concentrations (less than 30 mmol) hexoseY
H values were 25.5 for cellulose and 28.5 for cellobiose, while at hexose levels greater than 30 mmolY
H values were 13.5 and 15, respectively. Shifts in metabolism towards greater lactic acid production resulted in decreased ATP production; however, this did not cause early growth cessation, as these shifts occurred after the drop inY
H.Issued as NRCC No. 27409. 相似文献
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Hallberg BM Henriksson G Pettersson G Vasella A Divne C 《The Journal of biological chemistry》2003,278(9):7160-7166
The extracellular flavocytochrome cellobiose dehydrogenase (CDH; EC ) participates in lignocellulose degradation by white-rot fungi with a proposed role in the early events of wood degradation. The complete hemoflavoenzyme consists of a catalytically active dehydrogenase fragment (DH(cdh)) connected to a b-type cytochrome domain via a linker peptide. In the reductive half-reaction, DH(cdh) catalyzes the oxidation of cellobiose to yield cellobiono-1,5-lactone. The active site of DH(cdh) is structurally similar to that of glucose oxidase and cholesterol oxidase, with a conserved histidine residue positioned at the re face of the flavin ring close to the N5 atom. The mechanisms of oxidation in glucose oxidase and cholesterol oxidase are still poorly understood, partly because of lack of experimental structure data or difficulties in interpreting existing data for enzyme-ligand complexes. Here we report the crystal structure of the Phanerochaete chrysosporium DH(cdh) with a bound inhibitor, cellobiono-1,5-lactam, at 1.8-A resolution. The distance between the lactam C1 and the flavin N5 is only 2.9 A, implying that in an approximately planar transition state, the maximum distance for the axial 1-hydrogen to travel for covalent addition to N5 is 0.8-0.9 A. The lactam O1 interacts intimately with the side chains of His-689 and Asn-732. Our data lend substantial structural support to a reaction mechanism where His-689 acts as a general base by abstracting the O1 hydroxyl proton in concert with transfer of the C1 hydrogen as hydride to the re face of the flavin N5. 相似文献